Fungal infections, exemplified by oral and invasive candidiasis, cause considerable morbidity and mortality in an ever-increasing human population: the immunocompromised. Treatment of patients with fungal infections is severely hampered by the development of antifungal drug resistance. The goal of our research is to address this health need by discovering novel therapeutic agents that overcome clinical drug resistance by inhibiting a major cause of antifungal resistance, the drug efflux pump molecules in the fungal cell membrane. Lead compounds will be identified by high throughput screening (HTS) of the Molecular Libraries Small Molecule Repository (MLSMR) using a multiplex assay system developed by Professor Sklar at the University of New Mexico Center for Molecular Discovery (UNMCMD) and a panel of strains of the model yeast Saccharomyces cerevisiae, developed in our laboratory. The strains express a range of individual fungal efflux pumps that will be the targets for the proposed HTS. The specific aims are to: 1. Undertake a primary screen of the MLSMR using a multiplex flow cytometry assay: Proof of principle preliminary experiments has indicated that the assay will be applicable to the panel of yeast strains expressing efflux pumps from the following clinically relevant fungi: Candida albicans, Candida glabrata, Candida krusei and Cryptococcus neoformans. The yeast cell-based multiplex approach developed involves the use of three 'sentinel'strains in an HTS which measures efflux of the fluorescent pump substrate Nile Red and follow-up microtitre fluorescent assays with the other panel strains to identify pump inhibitors. 2. Screen the hits obtained with a panel of three secondary screens: The hits obtained in the primary screen will confirmed using secondary screens previously developed in our laboratory. The secondary assays will confirm that the targets are fungal efflux pumps, the extent of inhibitory activity, and elucidate the nature of the inhibitor/target interaction. The compounds identified will guide further research to develop drugs that overcome antifungal resistance. PUBLIC HEALTH RELEVANCE: Our long-term goal is to improve the treatment of patients with opportunistic fungal infections by discovering compounds that will act in combination with current antifungal drugs to overcome antifungal drug resistance.